There is a growing appreciation that posttranslational modifications, such as protein glycosylation, dramatically increase polypeptide complexity and function (Rudd et al., 2001 Science 291:2370-2376; Varki, 1993 Glycobiology 3:97-130; Dwek, 1996 Chem. Rev. 96:683-720; Roth, 2002 Chem. Rev. 102:285-303; Ritchie et al., 2002 Chem. Rev. 102:305-319; Zachara and Hart, 2002 Chem. Rev. 102:431-438; Bertozzi and Kiessling, 2001 Science 291:2357-2364; Kleene and Schachner, 2004 Nat. Rev. Neurosci. 5:195-208; Bucior and Burger, 2004 Curr. Op. Struct. Biol. 14:631-637; Schmidt et al., 2003 Trends Microbiol. 11:554-561). For example, almost all cell surface and secreted proteins are modified by covalently-linked carbohydrate moieties and the glycan structures on these glycoproteins have been implicated as essential mediators in processes such as protein folding, cell signaling, cell migration, fertilization, embryogenesis, neuronal development, hormone activity, and the proliferation of cells and their organization into specific tissues (Taylor and Drickamer, 2007 Curr. Opin. Cell. Biol. 19:572-577). In addition, overwhelming data supports the relevance of glycosylation in pathogen recognition, inflammation, innate immune responses, and the development of autoimmune diseases and cancer (Ohtsubo and Marth, 2006 Cell 126:855-867; Brockhausen, 2006 EMBO Rep. 7:599-604; Brown et al., 2007 Crit. Rev. Biochem. Mol. Biol. 42:481-515; Crocker et al., 2007 Nat. Rev. Immunol. 7:255-266; van Kooyk and Rabinovich, 2008 Nat. Immunol. 9:593-601). The importance of protein glycosylation is also underscored by the developmental abnormalities observed in a growing number of human disorders (known as Congenital Disorders of Glycosylation or CDGs), caused by defects in the glycosylation machinery (Freeze, 2007 Curr. Mol. Med. 7:389-396).
Almost all naturally occurring protein glycosylations can be classified as either N-glycosides whereby N-acetyl glucosamine is linked to the amide side chain of an asparagine, or as O-glycosides whereby a saccharide is linked to the hydroxyl of serine, threonine or tyrosine (Buskas et al., 2006 Glycobiology 16:113R-136R). The biosynthesis of N-linked oligosaccharides occurs in the endoplasmic reticulum (ER) and Golgi complex. In the ER, a dolichol-linked Glc3Man9GlcNAc2 oligosaccharide precursor is biosynthesized and transferred en bloc to an Asn-X-Ser/Thr sequon on newly synthesized polypeptides through the action of the multi-subunit oligosaccharide transferase complex (Dempski and Imperiali, 2002 Curr. Opin. Chem. Biol. 6:844-850; Weerapana and Imperiali, 2006 Glycobiology 16:91R-101R). Subsequent trimming and processing of the transferred oligosaccharide results in a Man3GlcNAc2 core structure, which is transported to the medial stacks of the Golgi complex where maturation of the oligosaccharide gives rise to extreme structural diversity (Ellgaard and Helenius, 2003 Nat. Rev. Mol. Cell Biol. 4:181-191; Helenius and Aebi, 2004 Annu Rev. Biochem. 73:1019-1049; Helenius and Aebi, 2001 Science 291:2364-9; Mast and Moremen, 2006 Methods Enzymol. 415:31-46); (Essentials of Glycobiology. 2nd edition. Varki et al., ed., Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009). The complexity of N-glycan structures is largely based on the cell-specific expression of a collection of glycosyl transferases that specify the extension of oligosaccharide structures onto the trimmed Man3GlcNAc2 core structure. The switch from structural uniformity in the ER to diversification in the Golgi complex coincides with a marked change in glycan function. In the early secretory pathway, the glycans have a common role in the promotion of protein folding, quality control, and certain sorting events. This is in contrast to their roles in the Golgi complex, in which they are modified to perform a wide spectrum of functions displayed by the mature glycoproteins.
The first enzyme needed for the biosynthesis of complex N-linked glycans is GlcNAcT-I, which adds a β(1-2)GlcNAc moiety to the core mannoside to produce a hybrid structure (Schachter, 2000 Glycoconj. J. 17:465-483; Chen et al., 2002 Biochim. Biophys. Acta 1573:271-279). Further processing of the resulting saccharide by mannosidases creates the precursor for GlcNAcT-II, which catalyzes the conversion of a hybrid structure into the precursor for complex N-glycan biosynthesis. Thus, each of the GlcNAc moieties of the resulting compound is converted into a complex bi-antennary structure. The formation of multi-antennary structures, require additional GlcNAc moieties that are added by GlcNAcT-IV, V and VI. Each of the resulting GlcNAc moieties can be extended into a complex oligosaccharide that can be quite diverse in structure. Furthermore, GlcNAcT-III can add a bisecting moiety that cannot be extended by further monosaccharide moieties. Collectively, the GlcNAcT's can create N-glycans that have as many as five branches (Schachter, 2000 Glycoconj. J. 17:465-483; Chen et al., 2002 Biochim. Biophys. Acta 1573:271-279). Finally, further diversification can take place by the addition of a core α(1-6)-fucoside to the GlcNAc moiety linked to the side chain of Asn. It is important to note that the action of one glycosyl transferase can preclude the action of another one and therefore only a subset of linkages performed by the GlcNAc transferases can occur on any one N-glycan. For example, GlcNAcT-V requires the prior activation by GlcNAcT-II. GlcNAcT-III and V are mutually exclusive as the action of one may inhibit the action of the other. The mode of GlcNAcT-VI is not well understood but may require prior branching by GlcNAcT-II and GlcNAcT-V.